TY - JOUR
T1 - Synthesis and characterization of positively charged tPA as a prodrug using a heparin/protamine-based drug-delivery system
AU - Liang, Jun F.
AU - Li, Yong T.
AU - Connell, Maureen E.
AU - Yang, Victor C.
PY - 2000
Y1 - 2000
N2 - Positively charged peptides [(Arg)7Cys] were successfully linked to tissue-specific plasminogen activator (tPA) using cross-linking agent N-succinimidyl 3-(2-pyridyldithio) propionate. Specific amidolytic activity of this tPA/(Arg)7Cys (termed modified tPA, mtPA) was 3900 IU/μg as compared to 5800 IU/μg of the parent tPA. Both activation of plasminogen with mtPA (Km = 2.7 mM-1) and tPA (Km = 1.1 mM-1) in a purified system followed Michaelis-Menten kinetics. In addition, (Arg)7Cys modification did not result in significant changes in the fibrin-binding ability of tPA, and mtPA still retained a response to fibrinogen similar to that of the parent tPA. Compared with tPA, mtPA showed much stronger heparin affinity, and the heparin/mtPA complex was stable in human plasma. The activity of mtPA in such a complex was inhibited by heparin, and, unlike tPA, the heparin/mtPA complex did not cause statistically meaningful depletion of plasminogen, fibrinogen, and α2-antiplasmin in plasma. Using the chromogenic and the in vitro clot lysis assay, it was demonstrated that the heparin-induced inhibition of the mtPA activity was easily reversed following the addition of an adequate amount of protamine. To enhance the clot-targeting efficiency of the heparin/mtPA complex further, anti-fibrin immunoglobulin (IgG) was conjugated to heparin via an end-point attachment of heparin to the sugar moieties in the Fc region of the IgG. Results show that the activity of mtPA could also be blocked by the heparin/anti-fibrin IgG conjugate. These findings suggest the applicability of the heparin/protamine delivery system to abort the potential bleeding risks associated with clinical use of tPA.
AB - Positively charged peptides [(Arg)7Cys] were successfully linked to tissue-specific plasminogen activator (tPA) using cross-linking agent N-succinimidyl 3-(2-pyridyldithio) propionate. Specific amidolytic activity of this tPA/(Arg)7Cys (termed modified tPA, mtPA) was 3900 IU/μg as compared to 5800 IU/μg of the parent tPA. Both activation of plasminogen with mtPA (Km = 2.7 mM-1) and tPA (Km = 1.1 mM-1) in a purified system followed Michaelis-Menten kinetics. In addition, (Arg)7Cys modification did not result in significant changes in the fibrin-binding ability of tPA, and mtPA still retained a response to fibrinogen similar to that of the parent tPA. Compared with tPA, mtPA showed much stronger heparin affinity, and the heparin/mtPA complex was stable in human plasma. The activity of mtPA in such a complex was inhibited by heparin, and, unlike tPA, the heparin/mtPA complex did not cause statistically meaningful depletion of plasminogen, fibrinogen, and α2-antiplasmin in plasma. Using the chromogenic and the in vitro clot lysis assay, it was demonstrated that the heparin-induced inhibition of the mtPA activity was easily reversed following the addition of an adequate amount of protamine. To enhance the clot-targeting efficiency of the heparin/mtPA complex further, anti-fibrin immunoglobulin (IgG) was conjugated to heparin via an end-point attachment of heparin to the sugar moieties in the Fc region of the IgG. Results show that the activity of mtPA could also be blocked by the heparin/anti-fibrin IgG conjugate. These findings suggest the applicability of the heparin/protamine delivery system to abort the potential bleeding risks associated with clinical use of tPA.
UR - http://www.scopus.com/inward/record.url?scp=4244025108&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=4244025108&partnerID=8YFLogxK
U2 - 10.1208/ps020107
DO - 10.1208/ps020107
M3 - Article
AN - SCOPUS:4244025108
SN - 1522-1059
VL - 2
SP - 59
EP - 67
JO - AAPS PharmSci
JF - AAPS PharmSci
IS - 1
ER -